Abstract 50: Anchored multiplex PCR for detection of gene rearrangements and mutations using next-generation sequencing.

Abstract

Abstract We describe a rapid targeted RNA-seq library construction method for the detection of gene fusions in cancer using next generation sequencing. The method involves ligation of half-functional sequencing adaptors to double strand cDNA, followed by two successive PCR reactions using hemi-nested gene-specific 3’ primers with the 5’ adaptor primer, and generates libraries ready for bi-directional sequencing. RNA extracted from formalin-fixed paraffin-embedded biopsies can be used directly without the need to deplete ribosomal RNA, non-target mRNA or genomic DNA. Importantly, the assay can be performed without the need to know the 5’ partner in a gene fusion event. The method can also be applied to genomic DNA for detection of gene mutations, insertions, deletions and copy number changes. The method is very flexible and can allow multiplexing of over 500 targets. We have designed a lung cancer translocation panel which includes 18 amplicons for detections of gene rearrangements involving ALK, ROS1 and RET. For targeted genomic DNA-seq, we have designed a small 96-amplicon panel including tumor hotspots and entire coding sequences of key tumor suppressor genes TP53 and PTEN, and a 623-amplicon panel for 370 exons of 18 tumor suppressor genes. Unlike conventional PCR-based method, this method generates libraries with random sequencing start sites and allows for direct sequencing without having to spiking in controls (e.g. PhiX) on those platforms where high starting site complexity is required, such as the Illumina Miseq sequencer. The assays generally achieve high on-target specificity (>90%), and are very cost-effective. The targeted RNA-seq assay has detected various gene rearrangements in known FISH-positive cases with previously unknown 5’ partners using our archived samples. The small 96-amplicon genomic DNA assay showed 100% of targeted bases having >100-fold coverage and 99.9% having >500-fold coverage with minimal optimization. In our first attempt of targeting 370 exons (626 amplicons, target size 52.3 kilobases), this assay achieved 97% of targeted bases having >100-fold coverage and 93% having >500-fold using ¼ of a Miseq sequencing run. Citation Format: Zongli Zheng, Boryana Zhelyazkova, Divya Panditi, Hayley Robinson, Jeffrey A. Engelman, John A. Iafrate, Long Le. Anchored multiplex PCR for detection of gene rearrangements and mutations using next-generation sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 50. doi:10.1158/1538-7445.AM2013-50